#METABOLOMICS WORKBENCH diwu_20230914_023703 DATATRACK_ID:4313 STUDY_ID:ST002875 ANALYSIS_ID:AN004712 PROJECT_ID:PR001797
VERSION             	1
CREATED_ON             	September 25, 2023, 9:28 am
#PROJECT
PR:PROJECT_TITLE                 	LAT1-4F2hc Lipidomics
PR:PROJECT_TYPE                  	MS identification
PR:PROJECT_SUMMARY               	The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino
PR:PROJECT_SUMMARY               	acids, hormones and drugs. Its dysfunction is associated with many cancers and
PR:PROJECT_SUMMARY               	immune/neurological disorders. Here, we apply native mass spectrometry
PR:PROJECT_SUMMARY               	(MS)-based approaches to provide evidence of super-dimer formation
PR:PROJECT_SUMMARY               	(LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we
PR:PROJECT_SUMMARY               	discover four endogenous phosphatidylethanolamine (PE) molecules at the
PR:PROJECT_SUMMARY               	interface and C-terminus of both LAT1 subunits. We find that interfacial PE
PR:PROJECT_SUMMARY               	binding is regulated by 4F2hc-R183 and is critical for regulation of
PR:PROJECT_SUMMARY               	palmitoylation on neighbouring LAT1-C187. Combining native MS with mass
PR:PROJECT_SUMMARY               	photometry (MP) we reveal that super-dimerization is sensitive to pH, and
PR:PROJECT_SUMMARY               	modulated by complex N-glycans on the 4F2hc subunit. Free LAT1 is also present
PR:PROJECT_SUMMARY               	in the lysosome after interferon-γ (IFN-γ) stimulation suggesting that the
PR:PROJECT_SUMMARY               	assembly of LAT1-4F2hc is regulated by redox dynamics in vivo. Together our
PR:PROJECT_SUMMARY               	results link PTM and lipid binding with regulation and trafficking of the
PR:PROJECT_SUMMARY               	LAT1-4F2hc super dimer.
PR:INSTITUTE                     	University of Oxford
PR:DEPARTMENT                    	Kavli Institute for Nanoscience Discovery
PR:LABORATORY                    	Prof. Carol Robinson Group
PR:LAST_NAME                     	Wu
PR:FIRST_NAME                    	Di
PR:ADDRESS                       	Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, OX1
PR:ADDRESS                       	3QU, UK
PR:EMAIL                         	di.wu2@chem.ox.ac.uk
PR:PHONE                         	+4401865275278
#STUDY
ST:STUDY_TITLE                   	LAT1-4F2hc Lipidomics
ST:STUDY_SUMMARY                 	The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino
ST:STUDY_SUMMARY                 	acids, hormones and drugs. Its dysfunction is associated with many cancers and
ST:STUDY_SUMMARY                 	immune/neurological disorders. Here, we apply native mass spectrometry
ST:STUDY_SUMMARY                 	(MS)-based approaches to provide evidence of super-dimer formation
ST:STUDY_SUMMARY                 	(LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we
ST:STUDY_SUMMARY                 	discover four endogenous phosphatidylethanolamine (PE) molecules at the
ST:STUDY_SUMMARY                 	interface and C-terminus of both LAT1 subunits. We find that interfacial PE
ST:STUDY_SUMMARY                 	binding is regulated by 4F2hc-R183 and is critical for regulation of
ST:STUDY_SUMMARY                 	palmitoylation on neighbouring LAT1-C187. Combining native MS with mass
ST:STUDY_SUMMARY                 	photometry (MP) we reveal that super-dimerization is sensitive to pH, and
ST:STUDY_SUMMARY                 	modulated by complex N-glycans on the 4F2hc subunit. Free LAT1 is also present
ST:STUDY_SUMMARY                 	in the lysosome after interferon-γ (IFN-γ) stimulation suggesting that the
ST:STUDY_SUMMARY                 	assembly of LAT1-4F2hc is regulated by redox dynamics in vivo. Together our
ST:STUDY_SUMMARY                 	results link PTM and lipid binding with regulation and trafficking of the
ST:STUDY_SUMMARY                 	LAT1-4F2hc super dimer.
ST:INSTITUTE                     	University of Oxford
ST:DEPARTMENT                    	Kavli Institute for Nanoscience Discovery
ST:LABORATORY                    	Prof. Carol Robinson Group
ST:LAST_NAME                     	Wu
ST:FIRST_NAME                    	Di
ST:ADDRESS                       	Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, OX1
ST:ADDRESS                       	3QU, UK
ST:EMAIL                         	di.wu2@chem.ox.ac.uk
ST:PHONE                         	+4401865275278
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:CELL_STRAIN_DETAILS           	HEK293F
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	LAT1-4F2hc_WT_1	Treatment:Untreated	RAW_FILE_NAME=LAT1-4F2hc_WT_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	LAT1-4F2hc_WT_2	Treatment:Untreated	RAW_FILE_NAME=LAT1-4F2hc_WT_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	LAT1-4F2hc_WT_delipidated_1	Treatment:Delipidated	RAW_FILE_NAME=LAT1-4F2hc_WT_delipidated_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	LAT1-4F2hc_WT_delipidated_2	Treatment:Delipidated	RAW_FILE_NAME=LAT1-4F2hc_WT_delipidated_2.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	The LAT1-4F2hc complex was buffer-exchanged into 1M ammonium acetate, pH 7.0
CO:COLLECTION_SUMMARY            	with 2 mM OGNG and incubated with 1 µg trypsin overnight at 37 °C. The
CO:COLLECTION_SUMMARY            	digested peptide/lipid mixture was dried using a SpeedVac vacuum concentrator
CO:COLLECTION_SUMMARY            	(Thermo Fisher Scientific)
CO:SAMPLE_TYPE                   	HEK cells
#TREATMENT
TR:TREATMENT_SUMMARY             	The LAT1-4F2hc complex was buffer-exchanged into 1M ammonium acetate (pH 7.0)
TR:TREATMENT_SUMMARY             	with 20 mM OGNG using a 100 kD MWCO centrifugal filter (Amicon Ultra-0.5 ml,
TR:TREATMENT_SUMMARY             	Millipore) and incubated for 4 h at room temperature with gentle mixing. Excess
TR:TREATMENT_SUMMARY             	lipomicelles and detergents were removed by buffer-exchanging into 1 M ammonium
TR:TREATMENT_SUMMARY             	acetate with 2 mM OGNG using the same filter.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The lipid mixture was reconstituted with 70% mobile phase A (acetonitrile/H2O:
SP:SAMPLEPREP_SUMMARY            	60/40, 10 mM ammonium formate and 0.1% formic acid) and 30% mobile phase B
SP:SAMPLEPREP_SUMMARY            	(isopropanol/acetonitrile: 90/10, 10 mM ammonium formate and 0.1% formic acid)
SP:SAMPLEPREP_SUMMARY            	for the following LC-MS/MS analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Column information: https://www.thermofisher.com/order/catalog/product/164568
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000
CH:COLUMN_NAME                   	Thermo Acclaim PepMap 100 C18 (150 x 0.007 mm, 3um, 100A)
CH:SOLVENT_A                     	60% acetonitrile/40% water; 10 mM ammonium formate; 0.1% formic acid
CH:SOLVENT_B                     	90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
CH:FLOW_GRADIENT                 	30%-99%
CH:FLOW_RATE                     	300 nl/min
CH:COLUMN_TEMPERATURE            	40
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo LTQ XL
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	-
MS:MS_RESULTS_FILE               	ST002875_AN004712_Results.txt	UNITS:Peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END